Cystic Fibrosis (CF) is a fatal recessive genetic disease characterized by abnormalities in chloride transport (McPherson & Dorner, 1991). The locus of the disease has been traced to mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). J. R. Riordan et al., Science (1989) 245:1066-1073; B. Kerem et al., Science (1989) 245:1073-1080. Correction of the underlying gene defect by complementation or replacement of the defective CFTR is the ultimate cure for CF. Gene therapy, the in vivo delivery and expression of genes, is a fast-developing science that can be used to replace defective genes.
Gene therapy is already feasible. T. Friedmann, Science (1989) 244:1275-1281; M. Bluestone, Biotechnol (1992) 10:132-134. Systems and polymers for delivery of polynucleotides are known in the art. P. L. Felgner, Adv Drug Delivery Rev (1990) 5:163-187. Adenoviral vectors have been used to transfer CFTR to the cotton rat lung in vivo. M. A. Rosenfeld et al., Cell (1992) 68:143-155. Although high levels of transfection in vivo have been reported with the adenoviral vectors, non-viral delivery systems have a number of advantages and should be vigorously developed. Rosenfeld et al., supra; M. A. Rosenfeld et al., Science (1991) 252:431-434.
During the past decade, a number of methods have been developed to introduce functional genes into mammalian cells in vitro. These techniques are applicable to gene therapy if the target cells can be removed from the body, treated, and the transfected cells amplified and then returned to the patient. This option is not possible for CF patients. At present the best in vivo transfection efficiencies are obtained with retroviruses (Bluestone, supra) and adenoviruses (Rosenfeld et al., supra). However the efficiency is variable and a concern is that virus based gene delivery might cause viral infection or cancer. Initial human clinical trials have revealed no acute complications of retroviral vectors but the possibility of long-term complications mandate careful patient monitoring. K. Cornetta et al., Human Gene Ther (1991) 2:3-14.
The risks of using viral based vectors and the conceptual advantages in using plasmid DNA constructs for gene therapy (discussed in P. L. Felgner et al., Nature (1991) 349:351-352) have led to a parallel development of various physical and chemical methods for gene transfer. The most intensely studied systems involve treatment of the cells with calcium phosphate or a cationic facilitator (Felgner et al., supra). Other popular methods involve DNA injection during physical puncture of the membrane (M. R. Capecchi, Cell (1980) 22:479-485) or passive uptake during permeabilization or abrasion of the cellular membrane (Felgner et al., supra). Each method is intrinsically aggressive and applicable only in vitro.
This invention is in the field of direct gene delivery that does not involve the use of viral vehicles. A non-viral carrier for gene delivery must be able to surmount many barriers: it must survive in circulation, it must be able to target the cell of choice, it must be able to introduce DNA into the cytoplasm, and it must be able to transport the DNA into the nucleus.
Masking. One concern about the direct delivery of genes in vivo is the ability of the polynucleotide to survive in circulation long enough to arrive at the desired cellular destination. "Masking", or protecting the polynucleotides is one way to address this concern.
Microparticulates (such as the erythrocyte ghost, reconstituted viral envelopes and liposomes) have been used in part as protection in gene transfer. C. Nicolau et al., Crit Rev Ther Drug Carr Sys (1989) 6:239-271; R. J. Mannio et al., Biotechniques (1988) 6:682-690. The most successful liposome system uses the cationic lipid reagent N-[1(-2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). P. L. Felgner et al., Proc Natl Acad Sci (USA) (1987) 84:7413-7417. DOTMA is mixed with phosphatidylethanolamine (PE) to form the reagent Lipofectin.TM.. The advantage of using Lipofectin.TM. is that the cationic liposome is simply mixed with the DNA and added to the cell. It is not necessary to encapsulate the DNA inside of the liposome with the cationic reagents. Lipofectin.TM. has been used to transfect reporter genes into human lung epithelial cells in culture (L. Lu et al., Pflugers Arch (1989) 415:198-203), to introduce the CAT gene into rats by intratracheal route (T. A. Hazinski et al., Am J Respir Cell Mol Biol (1991) 4:206-209) and to introduce the CAT gene into mice by the intratracheal and intravenous route (K. L. Brigham et al., Am J Med Sci (1989) 298:278-281; A. Bout et al., "Abstracts of the 1991 Cystic Fibrosis Conference", Abstract no. 87 (1991)). About 50% of the airway epithelial cells transiently expressed the .beta. galactosidase reporter gene (Hazinski et al., supra) but the level of expression was not quantitated. When chloramphenicol acetyltransferase (CAT) attached to a steroid sensitive promoter was transfected into rat lung, expression could be positively regulated by dexamethasone. Hazinski et al., supra. Cytotoxicity is a problem with high concentrations of Lipofectin.TM..
Substitutes for DOTMA include lipopolyamine (J. Loeffler et al., J Neurochem (1990) 54:1812-1815), lipophilic polylysines (X. Zhou et al., Biochim Biophys Acta (1991) 1065:8-14) and a cationic cholesterol (X. Gao et al., Biochem Biophys Res Comm (1991) 179:280-285). These have been used to mediate gene transfer in culture. Although there is some improvement over transfection rates observed with Lipofectin.TM. (about threefold), toxicity remains a problem. Studies on the mechanism responsible for transfection using the cationic lipids have been notably lacking. The past approach has been to synthesize different cationic lipids and try them in transfection assays, rather than to systematically study how the delivery systems introduce DNA into the cell. DOTMA/PE liposomes can undergo bilayer fusion with anionic liposomes (N. Duzgunes et al., Biochem (1989) 28:9179-9184) which suggests that direct entry of the DNA via the plasma membrane is involved with DOTMA's mode of action. High efficiency transfection using cationic lipids systems requires the inclusion of PE, possibly because PE can form intramembrane lipid intermediates which facilitate membrane fusion. The role of PE in membrane permeabilization and fusion has been extensively studied. E.g., M.-Z. Lai et al., Biochem (1985) 24:1646-1653; H. Ellens et al., Biochem (1986) 25:285-294; J. Bentz et al., Biochem (1987) 26:2105-2116).
Cellular Targeting. Efficient gene transfer requires targeting of the DNA to the cell of choice. Recently, procedures based upon receptor mediated endocytosis have been described for gene transfer. G. Y. Wu et al., J Biol Chem (1987) 262:4429; G. Y. Wu et al., J Biol Chem (1988) 263:14621-14624. A cell-specific ligand-polylysine complex is bound to nucleic acids through charge interactions. The resulting complex is taken up by the target cells. Wu et al., supra, reported efficient transfection of the human hepatoma cell line HepG2 and of rat hepatocytes in vivo using this delivery system with asialoorosomucoid as a ligand. Huckett et al., Biochem Pharmacol (1990) 40:253-263, reported stable expression of an enzymatic activity in HepG2 cells following insulin-directed targeting. Finally Wagner et al., Proc Natl Acad Sci (USA) (1990) 87:3410-3414 and (1991) 88:4255-4259 observed transferrin-polycation-mediated delivery of a plasmid into the human leukemic cell line K-562 and subsequent expression of the encoded luciferase gene. However, the described delivery systems are based upon high molecular weight targeting proteins linked to DNA through a polylysine linker. These large ligand-polycation conjugates are heterogenous in size and composition, not chemically well-defined, and difficult to prepare in a reproducible fashion (Wu et al., supra; Wagner et al., supra). Moreover, in many of the receptor mediated systems, chloroquine or other disruptors of intracellular trafficking are required for high levels of transfection. In one study, adenovirus has been used to enhance gene delivery of the receptor mediated systems. D. T. Curiel et al., Proc Natl Acad Sci (USA) (1991) 88:8850-8854.
Together these studies show that genes can be delivered into the interior of mammalian cells by receptor mediated endocytosis and a fraction of the exogenous DNA escapes degradation, enters the nucleus, and is expressed. The level of expression is low, probably due to the limited entry of the foreign DNA into the cytoplasm.
Charge Neutralization and Membrane Permeabilization. Direct delivery of genes is aided by the ability to neutralize the large negative charge on the polynucleotide, and the (often concomitant) ability to permeabilize the membrane of the targeted cell. The use of polycations to neutralize the polynucleotide charge and aid in the membrane permeabilization and translocation is well known. Felgner, supra. Cationic lipids have also been used for this purpose. P. L. Felgner et al., Proc Natl Acad Sci (USA) (1987) 84:7413-7417; U.S. Pat. No. 4,946,787 to Eppstein et al. Certain cationic lipids termed lipopolyamines and lipointercalants are also known. J.-P. Behr, Tet Lett (1986) 27:5861-5864.
Subcellular Localization. Once the polynucleotide has entered the targeted cell, direct delivery of genes would be aided by the ability to direct the genes to the proper subcellular location. One obvious target for the delivery of deoxyribonucleotides is the nucleus. Ligands known to aid in this process are nuclear localization peptides, or proteins containing these nuclear localization sequences. C. Dingwall et al., TIBS (1991) 16:478-481.
Y. Kaneda et al., Science (1989) 243:375-378, showed that the transfection efficiency obtained with reconstituted viral envelopes is increased when the foreign gene is co-delivered into the target cells with nuclear proteins. DNA mixed with nuclear proteins exhibit a modest increase in transfection over DNA that was mixed with albumin (Kaneda et al.). The assumption is that the DNA is incorporated into the nucleus more readily when proteins containing the nuclear localization sequence (NLS) SEQ ID NO:1 (P. A. Silver, Cell (1991) 64:489-497) are associated with the plasmid. The NLS on a protein designates it for transport through the nuclear pore. Nuclear localization sequences of 14 amino acids have been attached to a variety of macromolecules and even gold particles (150 A diameter) and, when introduced into the cytoplasm, they are rapidly incorporated into the nucleus (D. R. Findlay et al., J Cell Sci Supp (1989) 11:225-242; Silver, supra). The suggestion that nuclear entry is rate limiting for successful, stable transfection is also supported by the finding that plasmid DNA microinjected into the cytoplasm is unable to bring about transfection of cells (no transfection out of 1000 cytoplasmic injections, whereas microinjection of plasmids directly into the nucleus results in transfection in greater than 50% of the microinjected cells. Cappechi, supra. If the attachment of nuclear localization signals on the plasmid leads to transport of plasmid DNA into the nucleus, the transfection efficiency should increase. We propose a novel method to attach NLS and other ligands to the desired polynucleotide.
Finally, investigators have demonstrated that transfection efficiencies increase when DNA is condensed using various cationic proteins. T. I. Tikchonenko et al., Gene (1988) 63:321-330; M. Bottger et al., Biochim Biophys Acta (1988) 950:221-228; Wagner et al., supra. The reason why DNA condensation increases transfection is not readily apparent, it may increase cellular uptake of DNA (Wagner et al., supra) but it also can decrease susceptibility of the DNA to nuclease activity which may result in higher amounts of intact DNA in the cell.
Polynucleotide Association. Direct delivery of genes associated with one of the above-discussed classes of molecules, is further aided by the ability of those components to remain associated with the DNA. Wu et al., supra, associated their receptor ligand with the polynucleotide by covalently attaching the ligand to the polycation polylysine. Wagner et al., Bioconjugate Chem. (1991) 2:226-231, in addition to polylysine, also covalently attached the ligand to a DNA intercalator, ethidium homodimer (5,5'-diazadecamethylenebis(3,8-diamino-6-phenylphenanthridium) dichloride dihydrochloride). P. E. Nielsen, Eur J Biochem (1982) 122:283-289, associated photoaffinity labels to DNA by covalent attachment to 9-aminoacridine and certain bis-acridines.
None of the above references describe a system for polynucleotide delivery aimed at multiple aspects of the problems involved in bringing a circulating polynucleotide to a targeted subcellular location of a targeted cell. This invention addresses those problems by associating the polynucleotide with a combination of one or more of the following functional components: DNA-masking components, cell recognition components, charge-neutralization and membrane-permeabilization components, and subcellular localization components.